1. Field of the Invention
The present invention is generally related to digital pathology and, more particularly, to correcting the sensitivity of a group of similar fluorescence microscopy imaging instruments, as well maintaining the sensitivity of individual instruments over time and/or environmental variations.
2. Related Art
One frequently claimed benefit of fluorescence microscopy is the quantitative nature of signal intensity measurements. Unfortunately, this claimed benefit is commonly misunderstood to imply that fluorescent signals can easily be related back to some absolute intensity standard. In practice, absolute (calibrated) measurements are difficult, if not impossible, to achieve due to the lack of stable, calibrated reference standards.
Fluorescence microscopy system calibration, in terms of absolute sensitivity, requires the use of a calibrated reference standard. Due to difficulties of specimen preparation, and in particular, the unavoidable effect of sample bleaching over time, it is impossible to create a stable, organically based specimen suitable for calibration of a fluorescence microscope. Some stable, inorganic specimens are available, but due to variations in spectral response, these are not suitable for accurate calibration.
Unlike brightfield microscopy, fluorescence signal calibration is complicated by the fact that it is not simply a function of broadband optical transmission. It also has a strong relationship to wavelength. Fluorochromes have been designed to have very narrow spectral response, with their response curves overlapping the steep edges of multiple bandpass filters within the illumination and imaging paths. Even small variation in fluorochrome or filter bandwidths result in changes in the sensitivity of the system. For this reason, it is extremely difficult to predict the overall system sensitivity to a particular fluorochrome based on calibration of individual optical components.
Therefore, what is needed is a system and method that overcomes these significant problems found in conventional systems, as described above.